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Full text of "The Effects of a One-Tesla Magnet on Human Fibroblast Cell Growth"

Digitized by the Internet Archive 

in 2012 with funding from 

LYRASIS Members and Sloan Foundation 



http://archive.org/details/effectsofoneteslOOchap 



The Effects of a One-Tesla Magnet on Human Fibroblast Cell Growth 

by 
Ashley Chaplin 



A Thesis Submitted in Partial Fulfillment of 
Requirements of the CSU Honors Program 

for Honors in the degree of 

Bachelor of Science 

in 

Biology, 

College of Science, 

Columbus State University 







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Chaplin 1 

Abstract 

The controversy regarding the ill effects of electromagnetic fields began in 1979 
when Nancy Wertheimer and Ed Leeper claimed to have found a connection between 
childhood leukemia and power lines. In 1997, a group of researchers led by Martha 
Linet, M. D., attempted to provide evidence that there was no link between cancer and 
electromagnetic fields. The study showed that the risk of acute lymphoblastic leukemia 
did not escalate with increasing electromagnetic field levels in the children's homes. 
When compared to the Earth's static magnetic field of 0.5 Gauss, these fields were 
extremely small. Within the past decade, magnets have been used to treat various 
medical conditions including arthritis and migraines. Magnetotherapy is the term coined 
for this alternative approach to medicine which requires placing medical magnets on 
painful areas to reduce soreness and accelerate healing. The actual mechanism by 
which, and to what extent, magnets affect the body is unclear. Recent experiments 
involving Xenopus embryos indicate that huge magnetic fields of approximately 17 
Tesla 1 can change the second and third cleavage planes of development. These 
planes will orient, vertically or horizontally, to the direction of the applied magnetic field 
(Denegre, et. al. 1998). The potential effects of magnetic fields on the growth of human 
fibroblast cells were investigated in this study. Cell cultures were split and the new 
cultures were exposed to a one-TesIa magnetic field for approximately thirty-six hours 
during their growth phase. Half of the exposed cultures were counted for proliferation 
rate and the remainder of the cultures were analyzed for patterns of growth. An F test 
indicated that there was no significant difference in the growth rates between 
experimental and control cultures. A Chi square test was used to examine whether 
cells aligned themselves with the magnetic field during growth. The statistics showed 
that the data for the control and experimental groups were both significantly different 
from a random pattern. Since both the control and experimental groups had significant 
results, it can be concluded that the growth patterns of fibroblasts from the experimental 
group were no different than those in the control group. The results of this study 
indicate that magnets do not appear to have an effect on fibroblast growth rates or 
patterns. This work supports the contention that the reports of positive responses to 



1 One Tesla is equivalent to 10,000 Gauss. 



Chaplin 2 

magnetotherapy are due to a placebo effect. It also weakens the argument that 
electromagnetic fields cause cancer by increasing the growth rates of cells. 

Introduction 

Magnets, bipolar objects capable of attracting iron, steel, and other metallic 
items, have varying degrees of strength. The units of measurement for magnets include 
the Gauss and Tesla. These units are linked with the size of the magnet to determine 
its pulling power. Various materials are used to make magnets. Ferrite or ceramic 
magnets contain iron and barium. Neodymium magnets are composed of iron, boron 
and neodymium, a rare earth metal. The size of the magnet often determines the type 
of material used for its construction (Welcome to Magnets 4 Health, 2002). Magnets 
produce magnetic fields that are associated with electric fields. These fields surround 
all types of electrical equipment and appliances; they are present when the equipment 
is energized. In the case of an appliance, electric fields are present when it is plugged 
into the socket, regardless of whether or not it is in use. A magnetic field is absent until 
the equipment is turned on and the electric current is flowing (Kloepfer, 1993). These 
electromagnetic fields have been researched for many years to determine if they 
produce harmful or beneficial effects within the body. 

The study of the Earth's magnetosphere has been a difficult task for scientists. 
Within the past fifty years, the development of spacecraft has made it possible to 
determine the forces behind the magnetosphere of the Earth and of other planets 
(Cowley, 1996). The Earth's magnetosphere is formed from two central components: 
the Earth's magnetic field, generated by currents flowing in the Earth's core, and the 
solar wind, streaming outward from the Sun at 300-800 km/sec (Cowley, 1996). 
According to theoretical principles established by Sydney Chapman and Vincenzo 
Ferraro in the 1930s, magnetic fields are moved by flowing plasmas which are subject 
to bending and twisting. The same magnetic fields exert forces on the plasmas in order 
to resist these motions (Cowley, 1996). When the Chapman-Ferraro principles are 
applied to the Earth's magnetic field, the plasma of the solar wind is associated with the 
magnetic field produced by all of the planets; the plasma of the Earth is associated with 
the Earth's magnetic field. When the plasmas encounter each other, they form distinct 
regions separated by a thin boundary (Cowley, 1996). The Earth's magnetic field does 



Chaplin 3 

not continue beyond this boundary. It remains around the planet to form the 
magnetosphere that provides protection from the solar plasma. 

Within the magnetosphere, the magnetic North and South poles produce field 
lines along which charged particles are driven back and forth. The Van Allen radiation 
belts are two regions located within these lines. The belts surround the Earth and are 
composed of many charged particles that originated in the solar wind (The Earth's 
Magnetic Field, 2002). These particles produce the Northern and Southern Lights at the 
poles. Another interesting component of the Earth's magnetic field is the poles' ability to 
reverse direction. Evidence to support this theory includes the study of the magnetic 
fields of fossil rocks. These rocks show a specific magnetic orientation depending upon 
the time of formation. A third important characteristic of the Earth's magnetic field is it 
strength. The magnetic field is essential in protecting the Earth from solar wind and it 
deflects most harmful radiation. This field is utilized by organisms, such as birds, as an 
aid in migration. Many behavioral scientists have studied the natural migratory patterns 
of animals to determine how they use the magnetic field. In 1966, Wolfgang Wiltschko 
observed the effects of a magnetic field on caged birds during the time of normal 
migration. He altered the direction of magnetic south and found that the birds gathered 
in the new direction (Theoretical and Computational Biophysical Group, 2002b). 
Research on rainbow trout has determined the presence of magnetite-like particles in 
the brain. The cells located near these particles are connected to a nerve that is 
triggered during trout exposure to a magnetic field (Travis, 1997). Scientists at the 
University of Illinois have found a blue-light photoreceptor, identified as cryptochrome, 
which may explain how animals use the Earth's magnetic field in migration (Theoretical 
and Computational Biophysical Group, 2002a). This receptor is involved in controlling 
an animal's day-night rhythm. It is possibly the site of a neurochemical reaction that 
allows birds to use visual clues from the magnetic field to remain on course (Theoretical 
and Computational Biophysical Group, 2002a). Experiments indicate that light is 
essential for the bird to respond. Scientists have found that the light must have a short 
wavelength such as blue-green. If the light has a long wavelength, the bird cannot 
orient to the field (Theoretical and Computational Biophysical Group, 2002a). If the 
Earth's magnetic field disappears, many animals will not be able to navigate in their 
environments. 



Chaplin 4 

The controversy regarding the ill effects of electromagnetic fields (EMF's) began 
in 1979 when Nancy Wertheimer and Ed Leeper claimed to have discovered a 
connection between childhood leukemia and power lines. These researchers studied 
EMF exposures of Colorado children who died of cancer from 1950 to 1973. They 
reported that children residing in high exposure homes were two to three times more 
likely than those from low exposure homes to develop cancer, particularly leukemia 
(Pool, 1990). Scientists discovered several flaws within the study. The primary error 
was found in the method used to determine exposure to EMF's. Rather than measure 
the EMF strength, the scientists estimated the value by referring to wire code 
information that correlated with the types of power lines located near the homes. This 
information has been found to be an inaccurate indicator of actual EMF strength. Also, 
the study was not blind, because the researchers knew if cancer patients lived in the 
homes. David Savitz, at the University of Colorado Medical School, completed a 
cancer-EMF study in 1988. After he was asked to replicate Wertheimer's work, Savitz 
found similar results and used statistical analyses to support his conclusions. However, 
he was questioned due to the small sample size of his study. Other experiments by 
many researchers were completed, but the results varied from evidence supporting a 
possible link to the absence of a connection. The risk of cancer was associated 
primarily with children. Few studies had reported a correlation between adult cancer 
and exposure to power lines. Adults had an increased risk of cancer if they were 
electrical workers and spent long periods of time near high frequency EMF's. Many 
people are skeptical of this research. An article in Science suggests that if a link exists 
between EMF's and cancer, the connection should be supported by higher numbers of 
childhood leukemia cases due to the increase in electricity use of the past several 
decades (Pool, 1990). If 30-40% of childhood cancers are caused by EMF's, these 
types of cancers must have experienced a major increase during the last forty years. 
Epidemiologists do not agree on how cancer rates have changed over time, but it would 
be difficult for them to miss a large trend (Pool, 1990). In 1997, Martha Linet, M. D., and 
a group of researchers attempted to end the cancer-EMF debate. Their study, unlike 
previous research, included a large number of children. The controls were chosen 
based upon similarities to the cancer patients. Actual measurements of EMF's were 
taken in several rooms of the homes as well as in former residences. The technicians 



Chaplin 5 

who measured the fields were not informed of the health status of the residents (Linet 
et. al., 1997). The study showed that the risk of acute lymphoblastic leukemia did not 
escalate with increasing EMF levels in the children's homes (Campion, 1997). These 
levels were extremely small when compared to the Earth's static magnetic field of 0.5 
Gauss. Some scientists believe that the connection between EMF's and cancer 
possibly results from factors that have not been considered in the studies. Since high 
wire code homes are often located on heavily trafficked streets, air pollution may have a 
role in the leukemia link (Kaiser, 1996). Consequently, many researchers feel that 
studies on EMF's and cancer should be discontinued. More effort is needed to find 
definite origins and improved treatments of the disease. 

Despite the controversy surrounding EMF's, magnets have been used to treat 
various medical conditions including arthritis and migraines. Magnetotherapy is the 
term coined for this alternative approach to medicine. Medical magnets are placed on 
painful areas to reduce soreness and accelerate healing (Welcome to 
MagnetTherapy.com, 2002). Numerous companies have taken advantage of this new 
idea and mass production of magnets designed to aid all body parts has occurred. 
Jewelry, pillows, and mattresses containing magnets are now manufactured as well as 
body wraps and shoe insoles. Individual magnets are also produced for use on specific 
areas of the body. Different sizes and strengths are available for the consumer. One 
company, magnettherapy.com, sells anti-nausea wrist straps and anti-aging sleep 
masks in its "Unique and Unusuals" section. Magnets can be obtained for pets such as 
a magnetic pet bed and body wraps for horses. A new area of magnetic products 
includes items which magnetize food and water such as the Magnetic Water Muddler 
sold by Magnets 4 Health. 

While these companies have produced magnets for everyday use, medical 
professionals have developed several types of magnets for use in reducing bone loss 
and healing fractures. In the 1950s, scientists determined that bones are piezoelectric, 
indicating that bending or deforming the crystal structure creates local electric currents. 
Physiologists believe these currents explain why exercise strengthens bones and 
immobilization weakens them (Raloff, 1999). The OrthoLogic 1000 Bone Growth 
Stimulator is a portable, noninvasive machine worn by a patient for 30 minutes each day 
and it provides local magnetic field treatment (Orthologic, 2002). The company has had 



Chaplin 6 

a very high success rate with this FDA approved product. The OL1000 decreases a 
patient's need for surgery to correct a non-healing fracture by combining magnetic fields 
(dynamic and static) to speed bone growth. Another product, the SpinaLogic Bone 
Growth Stimulator, also uses these types of magnetic fields to aid a patient's healing 
process. New studies are experimenting with bone growth stimulators which can 
reduce osteoporosis. At Creighton University School of Medicine, a model has been 
produced which requires a person to stand on a platform twice a day for ten-minute 
sessions. During the sessions, the platform produces electric and magnetic fields. This 
treatment is anticipated to reduce bone loss (Raloff, 1999). These new machines and 
products that utilize EMF's as a means to improve health challenge previous evidence 
indicating that EMF's are damaging forces. 

The actual mechanism by which, and to what extent, magnets affect the body is 
unclear. Recent experiments involving Xenopus embryos indicate that magnetic fields 
of approximately 17 Tesla can change the second and third cleavage planes of 
development. These planes orient vertically or horizontally to the direction of the 
magnetic field. The magnets affect the position of the mitotic spindle apparatus. The 
researchers hypothesize that the magnetic field applies an additional torque to the 
spindle and astral microtubules (Denegre et. al., 1998). Few scientific experiments 
have discovered relationships between magnets and the human body. Many positive 
and negative reports of magnets have been recorded. People who believe magnets are 
beneficial claim the magnets increase blood circulation by dilating blood vessels and 
attracting charged particles (Anonymous, 1999a). The blood flow brings more oxygen 
and nutrients to the area and removes toxic wastes (Welcome to MagnetTherapy.com, 
2002). However, Robert Park, a physics professor at the University of Maryland, 
explains that magnets do not attract blood to a wounded area. Increased blood flow 
results in reddening of the skin, a symptom that is not evident in the presence of a 
magnet (Anonymous, 1999b). In association with increased circulation, magnets are 
also believed to align the water molecules in blood. John Schenck of the General 
Electric R&D Laboratory in Schenectady, New York states that no magnet exists which 
could arrange water molecules (Anonymous, 1999b). Other "benefits" of magnets 
include pH balance and increased hormone production such as melatonin. 



Chaplin 7 

Several negative effects have been associated with magnets. Researchers have 
found that EMF's interfere with drugs such as tamoxifen, which is used to prevent the 
reoccurrence of breast cancer. Robert P. Liburdy, a biologist at Lawrence Berkeley 
(Calif.) National Laboratory, claims that increased levels of EMF's inhibit tamoxifen's 
ability to decrease the growth rate of cancer cells in test tubes. He also states that 
EMF's affect the ability of melatonin to stop the growth of breast cancer cells (Raloff, 
1997). Some scientists believe that EMF's can increase the risk of cancer, because 
they cause susceptible cells to undergo increased replication, a characteristic of cancer 
cells. A study at Michigan State University indicates that immature red blood cells 
exposed to a low frequency EMF do not mature and replicate repeatedly (Sivitz, 2000). 
This experiment along with other contradictory studies indicates the need for further 
research which will determine the true effects of magnets on the body. 

Due to the controversy surrounding magnets and human health, I chose to 
research the effects of magnets on human fibroblast growth. I determined the influence 
of a strong magnet by growing cell cultures and studying the proliferation rate after a 
specific time period. I examined the pattern of development by staining the cells and 
describing the general direction of growth. Though my project did not provide answers 
to every question about magnetic fields, the results answered basic questions about the 
effects of magnets. 

Materials and Methods 

930 ml Dulbecco's Modified Eagles Medium with L-Glutamine 

100 ml Fetal Bovine Serum 

10 ml Antibiotic/Anti mycotic 

1750 ml Calcium and Magnesium Free Phosphate Buffered Saline (CMF-PBS) 

110mlTrypsin/EDTA 

160 culture flasks 

9 ml Trypan blue solution 

270 ml Wright/Giemsa stain solution 

270 ml absolute methanol 

270 ml phosphate buffer solution (pH = 6.9) 

270 ml distilled water 



Chaplin 8 

76 culture tubes for centrifuge 
2 Incubators 
Inverted microscope 
Light microscope 
1 Centrifuge 

1 Hemocytometer 
70% ethanol solution 

Sterile pipets: 5 and 10 ml; pipetors 
27 1-Tesla magnets 

2 vials of human fibroblast cells 
Duct tape 

Clothespins 

Several meters of 60 lb picture wire 

Pieces of wood (fir) 

Wire cutters 

Saw 

Sheet metal screws 

Drill 

Procedures were the same for each set of fibroblast cells. 23 days were required 
to study the proliferation rates and growth patterns of the cells. A 1-Tesla magnet was 
placed under each experimental flask. The control group and the experimental group 
were positioned in separate incubators. 

Proliferation Rates and Growth Patterns 

On Day 1, the Dulbecco's Modified Eagles Medium (supplemented with L- 
Glutamine, 10% fetal bovine serum, and 1% antibiotic/antimycotic) was prepared and 
labeled DMEM++. Two vials of human fibroblast cells were thawed in a 37°C water 
bath. The vials were dipped in alcohol before being transferred to the work area under 
the cell culture hood. For this procedure, five flasks and 20 ml of DMEM++ were 
needed. The contents of the vials were placed in one flask using sterile pipets. 20 ml of 
the medium were added. The cells were mixed thoroughly with the medium using the 



Chaplin 9 

pipets. 5 ml were transferred to each new flask. Two flasks were used as the source of 
the control group. The cultures were maintained in a water-jacketed CO2 incubator held 
at 37°C and 5% C0 2 . 

On Days 7, 14, and 21 , 1:3 splits were performed (refer to Appendix A for 
splitting procedure). The cultures were fed DMEM++ every 7 days beginning on Day 4 
(refer to Appendix A for feeding procedure). The cultures were checked for growth and 
contamination each day of the experiment. 

After the split on Day 21, 27 flasks were randomly selected for the experimental 
group and the remaining 27 flasks were used for the control group. The magnets were 
positioned in the incubator that contained the experimental group (refer to Appendix B 
for magnet placement [Figure 1]). The magnets were placed such that the central axis 
of the magnetic field was down the longitudinal center of the culture flask. The flasks 
were returned to the incubators. 

After 36 hours of growth, the flasks were removed from the incubators. 26 flasks 
(13 control, 13 experimental) were stained. These flasks were used to determine the 
growth patterns of the fibroblasts. The cells in 28 flasks (14 control, 14 experimental) 
were counted. 

Refer to Appendix C for staining procedure. 

Refer to Appendix D for counting procedure. 

Refer to Appendix E for procedure for determining growth patterns. 

Statistical Analysis 

An F-Test Two-Sample for Variances was used to evaluate the proliferation rates 
of viable and nonviable cells for the control and experimental groups. A Chi square test 
for each group determined the significance of the observed growth patterns. 

Results 

The cell count for the control group indicated that 189 viable cells and 86 
nonviable cells were present. The cell count for the experimental group indicated that 
197 viable cells and 120 nonviable cells were present. The percentage of viable to 
nonviable cells for the control group was 69% to 31%. The percentage of viable to 
nonviable cells for the experimental group was 62% to 38%. These values can be 



Chaplin 10 

found in Table 1. The data in Table 2 indicate the observed growth patterns for the 
control and experimental groups. 

For a statistical analysis of the proliferation rates, the F-Test Two-Sample for 
Variances (DF = 6) indicated that the critical value was 5.82. The calculated F value for 
the viable cells was 2.86 (Table 3). The calculated F value for the nonviable cells was 
2.94 (Table 4). 

For a statistical analysis of the observed growth patterns (refer to Appendix F for 
pictures of control and experimental cells [Figures 2, 3]), the degrees from Table 2 were 
combined according to the values in Table 5. For example, the number of cells 
observed from 30-39° was combined with the number of cells observed from 120-129°. 
Tables 6 and 7 contain this data. These tables also show the number of cells which 
cross only the y-axis. 

For evaluation of the observed growth patterns of the control and experimental 
groups, a Chi square analysis (DF = 8) was used. The critical X 2 value was 15.5. 
Tables 8 and 9 contain the data for the cells located on both axes: x and y. The 
calculated X 2 value for the control group was 96.3 (Table 8). The calculated X 2 value 
for the experimental group was 83.1 (Table 9). Tables 10 and 1 1 contain the data for 
the cells which cross only the y-axis. The calculated X 2 value for the control group was 
61.6 (Table 10). The calculated X 2 value for the experimental group was 72.2 (Table 
11). 



Chaplin 11 



Table 1. Viable and Nonviable Cell 

Counts of Control and Experimental 

Groups 


I Control 


Experimental 


Viable 


189 


197 


Nonviable 


86.0 


120 


Percent viable 


69.0% 


62.0% 


Percent nonviable 


31.0% 


38.0% 



Table 2. Observed Growth Patterns 


Degree (°) 


Control 


Experimental 


0-9 


1.000 


2.000 


10-19 


8.000 


8.000 


20-29 


35.00 


52.00 


30-39 


72.00 


80.00 


40-49 


84.00 


79.00 


50-59 


56.00 


116.0 


60-69 


56.00 


92.00 


70-79 


65.00 


82.00 


80-89 


54.00 


82.00 ! 


90-99 


72.00 


107.0 


100-109 


58.00 


78.00 


110-119 


57.00 


89.00 


120-129 


73.00 


86.00 


130-139 


68.00 


92.00 


140-149 


52.00 


41.00 


150-159 


35.00 


24.00 


160-169 


7.000 


3.000 


170-179 


4.000 


1.000 


Total 


857.0 


1114 | 



Table 3. F-Test Two-Sample 

for Variances 

Viable Cells 

a = 0.05 




Control 


Experimental 


Mean 


27.0 


28.1 


Variance 


40.7 


116 


Observations 


7.00 


7.00 


Degrees of 
Freedom 


6.00 


6.00 


Calculated F Value 


2.86 


Critical F Value 


5.82 


^gjgppj^l^l Critical > Calculated 



Table 4. F-Test Two-Sample 

for Variances 

Nonviable Cells 

a = 0.05 




Control 


Experimental 


Mean 


12.3 


17.1 


Variance 


26.9 


79.1 


Observations 


7.00 


7.00 


Degrees of 
Freedom 


6.00 


6.00 


Calculated F Value 


2.94 


Critical F Value 


5.82 


<v ^ J - | Critical > Calculated 



Chaplin 12 



Table 5. Degree (°) Combination 


Combined ° 


New° 


0-9 


90-99 


0-9 


10-19 


100-109 


10-19 


20-29 


110-119 


20-29 


30-39 


120-129 


30-39 


40-49 


130-139 


40-49 


50-59 


140-149 


50-59 


60-69 


150-159 


60-69 


70-79 


160-169 


70-79 


80-89 


170-179 


80-89 



Table 6. Observed Growth Patterns 
of the Control Group 


Degree (°) 


Number of Cells 


X-, Y- Axes 


Y- Axis ! 


0-9 


73.0 


31.0 


10-19 


66.0 


30.0 


20-29 


92.0 


38.0 


30-39 


145 


69.0 


40-49 


152 


71.0 


50-59 


108 


39.0 


60-69 


91.0 


36.0 


70-79 


72.0 


24.0 


80-89 


58.0 


25.0 


Total 


857 


363 



Table 7. Observed Growth Patterns 
of the Experimental Group 


Degree (°) 


Number ol 


F Cells 


X-, Y- Axes 


Y- Axis 


0-9 


109.0 


35.00 


10-19 


86.00 


37.00 


20-29 


141.0 


64.00 


30-39 


166.0 


100.0 


40-49 


171.0 


70.00 


50-59 


157.0 


71.00 


60-69 


116.0 


42.00 


70-79 


85.00 


48.00 


80-89 


83.00 


33.00 


Total 


1114 


500.0 



Table 8. Chi Square Analysis of 

Growth Patterns 

Control Group 

X-, Y- Axes 

Degrees of Freedom = 8 


Degree (°) 


Observed 


Expected 


(O - E) 2 /E 


0-9 


73.0 


95.2 


5.18 


10-19 


66.0 


95.2 


8.96 


20-29 


92.0 


95.2 


0.108 


30-39 


145 


95.2 


26.1 


40-49 


152 


95.2 


33.9 


50-59 


108 


95.2 


1.72 


60-69 


91.0 


95.2 


0.185 


70-79 


72.0 


95.2 


5.65 


80-89 


58.0 


95.2 


14.5 


Total 


857 


857 


96.3 


Calculated X 2 


96.3 


Critical X 2 


15.5 


q/jnlffiW&ii: Calculated > Critical 







Chaplin ' 


13 


Table 9. Chi Square Analysis 

Growth Patterns 

Experimental Group 

X-, Y- Axes 

Degrees of Freedom = 8 


of 


Degree (°) 


Observed 


Expected 


(O - E) 2 /E 


0-9 


109.0 


123.8 


1.770 


10-19 


86.00 


123.8 


11.50 


20-29 


141.0 


123.8 


2.390 


30-39 


166.0 


123.8 


14.40 


40-49 


171.0 


123.8 


18.00 


50-59 


157.0 


123.8 


8.900 


60-69 


116.0 


123.8 


0.4910 


70-79 


85.00 


123.8 


12.20 


80-89 


83.00 


123.8 


13.40 


Total 


1114 


1114 


83.10 


Calculated X 2 


83.10 


Critical X 2 


15.50 



Calculated > Critical 



Table 10. Chi Square Analysis of 

Growth Patterns 

Control Group 

Y- Axis 

Degrees of Freedom = 8 


Degree (°) 


Observed 


Expected 


(O - E) 2 /E 


0-9 


31.00 


40.33 


2.160 


10-19 


30.00 


40.33 


2.650 


20-29 


38.00 


40.33 


0.1350 


30-39 


69.00 


40.33 


20.40 


40-49 


71.00 


40.33 


23.30 


50-59 


39.00 


40.33 


0.04390 


60-69 


36.00 


40.33 


0.4650 


70-79 


24.00 


40.33 


6.610 


80-89 


25.00 


40.33 


5.830 


Total 


363.0 


363.0 


61.60 


Calculated X 2 


61.60 


Critical X 2 


15.50 




Calculated > Critical 



Table 11. Chi Square Analysis of 

Growth Patterns 

Experimental Group 

Y- Axis 

Degrees of Freedom = 8 


Degree (°) 


Observed 


Expected 


(O - E) 2 /E 


0-9 


35.00 


55.56 


7.610 


10-19 


37.00 


55.56 


6.200 


20-29 


64.00 


55.56 


1.280 


30-39 


100.0 


55.56 


35.50 


40-49 


70.00 


55.56 


3.750 


50-59 


71.00 


55.56 


4.290 


60-69 


42.00 


55.56 


3.310 


70-79 


48.00 


55.56 


1.030 


80-89 


33.00 


55.56 


9.160 


Total 


500.0 


500.0 


72.20 


Calculated X 2 


72.20 


Critical X 2 


15.50 



Calculated > Critical 



Chaplin 14 

Discussion 

When the fibroblasts were studied for proliferation rates, more viable and 
nonviable cells were found in the experimental group than the control group. The cell 
counts for the experimental and control groups were converted to percentages in order 
to compare the data (Table 1). The percentage of viable to nonviable cells was 
approximately the same for each group. An F-Test Two-Sample for Variances 
determined if the observed differences in proliferation rates were significant. This test 
indicated that the experimental group did not have a significantly higher or lower growth 
rate of viable cells compared to the control group (Table 3). Analysis of the nonviable 
cells with the F test provided similar results (Table 4). The number of nonviable cells in 
the experimental group was not significantly different than the control group. The 
magnets did not seem to have an effect on the proliferation rates of fibroblasts. The 
results support the study completed by Martha Linet et. al. in 1997. The data dispute 
the possible link between EMF's and cell proliferation as seen in cancer. This link is 
based upon the belief that EMF's cause cells to replicate repeatedly. The results also 
indicate that magnetic fields do not have an effect on fibroblast cell growth and further 
demonstrate that magnets of low power are unlikely to be cancer causing agents. 
However, studies do indicate that prolonged exposure to high levels of EMF's can 
increase an adult's cancer risk (Pool, 1990). The findings of this research dispute the 
beneficial effects of magnetotherapy. The absence of a change in proliferation rates 
shows that magnets probably do not have healing properties as they cannot increase 
the number of cells needed to repair a wound nor can they decrease the growth rate of 
harmful cells, such as tumor cells. 

When growth patterns were initially analyzed, Chi square tests for the cells 
located on both axes were completed. This analysis included all of the angles (0°- 
179°). The calculated X 2 values of the control and experimental groups were much 
greater than the critical X 2 values (data not shown). In order to minimize the difference 
between these results, the degrees of growth were combined according to the values 
presented in Table 5. For example, cells growing between and 9 degrees on the y- 
axis were equivalent to cells growing between 90 and 99 degrees on the x-axis. A 
second set of Chi square tests was completed on the combined data. For both control 
and experimental groups, the results indicated that the calculated X 2 values were 



Chaplin 15 

greater than the critical X 2 values (Tables 8, 9). The null hypothesis was rejected for 
both groups. Cell growth occurred in specific angles rather than random patterns. The 
importance of examining the experimental group as well as the control group was 
shown in this portion of the analysis. If the data for the experimental group were the 
only information to be statistically tested, the results would illustrate that there was a 
significant difference in growth patterns. It would be concluded that the magnets had an 
effect on cell growth. However, the Chi square test also indicated that there was a 
significant difference in growth patterns for the control group. Since null hypotheses 
were rejected for both groups, the results provided evidence that magnets did not have 
an effect on the growth patterns of fibroblasts. The control group showed that the cells 
typically grew in specific angles in the absence of magnets. 

As a final analysis, a Chi square test was applied to the cells which were present 
along the y-axis. The basis for this test was the placement of the magnets on the 
flasks. A magnet was aligned with the x-axis of each flask. If the magnets were to 
influence the growth of the fibroblasts, the cells on the y-axis would be affected more 
strongly than the cells along the x-axis. This is due to the magnetic field lines that enter 
and leave the magnets at the poles. These lines form a sphere. However, fibroblasts 
grow in a monolayer i.e. the cells do not grow on top of each other. Fibroblasts located 
along the y-axis are subjected to more magnetic field lines than the fibroblasts located 
along the x-axis, because the latter group is aligned with the magnet. The results of the 
Chi square test indicated that the calculated X 2 values were greater than the critical X 2 
values for the control and experimental groups (Tables 10, 11). The null hypotheses 
were rejected, illustrating that there was a significant difference in the growth patterns of 
both groups. Certain angles were observed more than others. This is most likely due to 
the fibroblasts' tendency to develop in groups. When the cells divide, the new cells 
often grow in the same direction as the original cells. Clumps of individual fibroblasts 
growing at the same angle were observed in the flasks. Since the control group 
experienced the same results as the experimental group, the data demonstrated that 
the magnets did not cause the observed growth patterns. Studies ofXenopus embryos 
have indicated that high power magnets can cause cells to grow in specific directions 
(Denegre et. al., 1998). These magnets of 17 Tesla are extremely powerful when 



Chaplin 16 

compared to the 1 Tesla magnets of this research. Inducing changes in the growth 
patterns of fibroblasts may be possible only in the presence of high power magnets. 

This study demonstrates that magnets do not have an effect on proliferation rates 
or growth patterns of fibroblasts. The research weakens the argument that EMF's and 
magnets increase the risk of cancer by causing vulnerable cells to replicate 
uncontrollably. No significant difference between proliferation rates of experimental and 
control cells is observed. Since the magnets are not influencing the cell cycle, they are 
not predisposing the cell to become cancerous. The study also supports previous 
research which finds that magnets do not cause biological events such as increased 
blood flow. This analysis of fibroblasts provides evidence that the reports of positive 
responses to magnetotherapy are possibly due to a placebo effect. However, it should 
be considered that these results are subject to change with the use of a higher power 
magnet as indicated by the work of Denegre et. al. 

Acknowledgments 

I would like to thank Dr. Glenn Stokes for his advice and assistance throughout 
the course of this project, Dr. William Birkhead for his helpful comments, and my 
parents, David and Kathy Chaplin, for their support. 



Literature Cited 

Campion, E.W. 1997. Power lines, cancer, and fear. New England Journal of Medicine 
337:44-46. 

Chen, I. 1999. An attractive way to ease pain?. Health 13:64-6. 

Cowley, S.W.H. 1996. The earth's magnetosphere. American Geophysical Union 8:9- 

16. 
Denegre, J.M., Valles, J.M. Jr., Lin, K., Jordan, W.B. and K.L. Mowry. 1998. Cleavage 

planes in frog eggs are altered by strong magnetic fields. Proceedings of the 

National Academy of Sciences 95: 1 4729-1 4732. 



Chaplin 17 

University of Tennessee. The earth's magnetic field. 

< http://csep10.phys.utk.edu/astr161/lect/earth/magnetic.html > (accessed April 

2002). 
Kaiser, J. 1996. Panel finds emfs pose no threat. Science 274:910. 
Kimberly Health Products. North pole magnets help the body heal itself. 

< http://vwvw.pain-relief-magnets.com > (accessed April 2002). 
Kloepfer, R.J. 1993. Are electric and magnetic fields a cause for concern?. Journal of 

Environmental Health 55:53. 
Linet, M.S., Hatch, E.E., Kleinerman, R.A., Robison, L.L., Kaune, W.T., Friedman, D.R., 

Severson, R.K., Haines, CM., Hartsock, C.T., Niwa, S., Wacholder, S. and R.E. 

Tarone. 1997. Residential exposure to magnetic fields and acute lymphoblastic 

leukemia in children. New England Journal of Medicine 337:1-7. 
[Anonymous]. 1999a Jul/Aug. Magnet therapy. WE Magazine 3:87 . 
[Anonymous]. 1999b Sept 27. Magnet therapy: what's the attraction?. Pain Weekly p9. 
OrthoLogic. 2004. Orthologic: leading the way to a bright future in orthobiologics. 

< http://www.orthologic.com > (accessed April 2002). 
Pool, R. 1990. Is there an emf-cancer connection?. Science 249:1096-1098. 
Raloff, J. 1997. Magnetic fields can diminish drug action. Science News 152:342. 
Raloff, J. 1999. Medicinal emfs. Science News 156:316-318. 
Sivitz, L. 2000. Cells proliferate in magnetic fields. Science News 158:196-197. 
Theoretical and Computational Biophysical Group. 2000. Light receptor may be key in 

how animals use Earth's magnetic field. 

< http://www.ks.uiuc.edu/Research/Press/GeoField.shtml > (accessed April 2002). 
Theoretical and Computational Biophysical Group. 2004. The magnetic sense of 

animals. < http://www.ks.uiuc.edu/Research/magsense/ms.html > (accessed April 

2002). 
Travis, J. 1997. Resolving the magnetoreception puzzle. Science News 152:365. 
Magnets4Health. Magnets4Health: the home of magnetic therapy. 

< http://www. magnets4health. co. uk/home. cfm > (accessed April 2002). 
Welcome to MagnetTherapy.com. < http://www.magnettherapy.com > (accessed April 

2002). 



Chaplin 18 

Appendix A 

Use a sterile technique for cell splitting and feeding. 
Procedure for Cell Splitting (1:3): 

1. Gather the supplies required for cell splitting: DMEM++, CMF-PBS (buffer), Trypsin, 
three culture flasks, sterile pipets, waste beaker. 

2. Use a 70% ethanol solution to sterilize the work area under the cell culture hood. 

3. Sterilize the different bottles before placing them in the work area. 

4. Using an aseptic technique, open the culture flask which contains the cells. Pour the 
medium into a waste beaker. 

5. Pipet 5ml of CMF-PBS into the flask and swirl it carefully before emptying the buffer 
into the beaker. 

6. Pipet 1ml of Trypsin into the flask. Swirl the flask and place it flat on the counter. 
Allow approximately ten minutes for the cells to detach from the bottom of the flask. 
Before completing the next step, the flask should be examined to ensure that the cells 
are no longer attached to the flask. 

7. Pipet 1 5ml of fresh medium into the flask. Use the pipet to draw up the liquid and 
spray the cell side of the flask to break the cell monolayer. Spray the side several 
times. 

8. Use the pipet to collect 5ml of the medium and place the liquid in one new flask. 
Repeat with the remaining 10ml and two other flasks. 

9. Return the flasks to the incubator and loosen the caps of the flasks. 

Procedure for Culture Feeding: 

1 . Follow steps 1- 5 as described in the procedure for cell splitting excluding the trypsin 
and three flasks. 

2. After emptying the buffer into the beaker, pipet 5ml of fresh medium into each flask. 

3. Return the flasks to the incubator and loosen the caps. 



Appendix B 
Magnet Placement: 



The magnets were attached to the incubator shelves using duct tape, 60 lb 
picture wire, clothespins, and pieces of wood (fir). 9 magnets were placed on each 
shelf in 3 rows. The duct tape was used to position the magnets. The wire was looped 



Chaplin 19 

through holes in the shelves to stabilize each magnet. Clothespins were disassembled 
and the wooden portions were placed under the wire that looped over the magnet. Not 
all magnets needed this treatment. Small pieces of fir were cut to fit the width of the 
shelves. Each piece was placed against the magnets and notched if necessary. 
Screws attached the wood to the shelves. 



►At 



, fed Pt: ■ . ^3 h- ?3JJp=S? 



~t 



n it 

set ats 







Figure 1. Magnet Placement on Incubator Shelf 



Appendix C 
Procedure for Cell Staining: 

1 . Open the flask and pour the medium into the waste beaker. 

2. Pipet 5 ml of CMF-PBS into the flask, swirl it, and empty the buffer into the waste 
beaker. 

3. Add 5 ml of absolute methanol to the flask so the layer of cells is covered. Allow the 
flask to sit for 10 minutes. Cover the flask so the alcohol does not evaporate. 

4. Pour the alcohol into the waste beaker and allow the cells to dry. 

5. Add 5 ml Wright/Giemsa stain solution to the flask so the entire layer of cells is 
covered. Leave the solution on the cells for 30 seconds and then empty it into the 
beaker. 



Chaplin 20 

6. Add 5 ml of buffer solution to the flask to cover the cells. Allow the buffer to sit for 3- 
8 minutes. Pour off the buffer and rinse the cells with 5 ml of distilled water. Allow cells 
to dry completely. 

7. Leave the cells in the flask and view them under a light microscope at 400X and 
1000X (oil immersion). 

Appendix D 
Procedure for Cell Counting: 

1 . Open the flask and empty the medium into a waste beaker. Pipet 5ml of CMF-PBS 
into the flask and rinse the cells. Pipet 1 ml of Trypsin into the flask and allow it to sit for 
10 minutes. Examine the flask under a microscope to ensure the cells have detached 
from the sides of the flask. 

2. Add 10ml of CMF-PBS and use it to spray the cell side of the flask to break the 
monolayer. Spray several times. 

3. Pour the cell suspension into a sterile culture tube. Repeat steps 1 - 3 for all flasks. 

4. Centrifuge the tubes at 500 g for 3 minutes. 

5. Use a pipet to remove the supernatant from the tube. The liquid can be discarded. 
Use another pipet to add 5ml of CMF-PBS to the tube to resuspend the pellet of cells. 
Mix the buffer and cells gently. Combine the contents of two tubes. 

6. Remove 0.2ml of the suspension and place it into a clean culture tube. Add 0.3ml of 
CMF-PBS and mix. 

7. Add 0.5ml of trypan blue solution and mix thoroughly using the pipet. The cells are 
now diluted at a 1:5 ratio (a dilution factor of 5). 

8. Place a coverslip on a hemocytometer. Use a pipet to transfer a small amount of the 
solution to both chambers. Place the pipet at the edge of the coverslip and slowly fill 
the chambers. Do not overfill or underfill. 

9. Choose a starting chamber and count all of the cells (living and dead) located in the 
center square and four corner squares of the hemocytometer. Keep a separate count of 
dark (nonviable) and light (viable) cells. 

10. Repeat this process on the remaining counting chamber of the hemocytometer. 



Chaplin 21 

Mathematical Procedure for Cell Count: 

1 . Choose one of the five squares in the counting chamber and count all of the cells 
within the square. For cells located along the edges, count the cells at the top and right 
edge lines. Repeat for all five squares in both chambers. 

2. Each square represents a total volume of 0.1 mm 3 (lO^cm 3 ). One cubic centimeter 
equals one millimeter. 

Formulas: 

Cell concentration 

Cells/ml = [(Total number of cells)/(Number of squares)] X dilution factor X 10 4 
Total number of cells in the culture 

Total cells = [(Number of cells)/(ml)] X original volume of cell suspension 
Percentage viability 

% viability = [(Number of viable cells)/(Total number of cells counted)] X 100 

Appendix E 
Procedure for Determining Growth Patterns: 

Pictures were taken of 26 flasks (13 control, 13 experimental). A grid was placed 
on each picture to divide it into four quadrants. The cells that were located along each 
axis were measured with a protractor to determine the angle of growth. 



Chaplin 22 



Magnet 
(1 Tesla) 



Cells 







Y-Axis 




\ 
► 


■ \ 






/ 


\ 


\ 


\ 


x v 


\ / / 




/ 


\ 


\ 


/ 


/ 










\ 


\ 


X-Axis 





Magnetic 
Field Lines 




Chaplin 23 



Appendix F 




• *i jft»i % - * * * 
1 . • ; % •• ♦ V 



i ♦ i • • %\\ 



" — V.. - ' '* 

■ 



V I 






* - • ' . \ -s 

«* ' * 'St**'" 



I . ■ .. • 



r> * 



• .*»./ 



•■ J i 



> 



9 % m 



./ : *« 






% „ 






• • • %% 












# * 




. ' .// 









r^rr- 



* • • ' #t • . * ■•* 

v •' *v %,/ % . *% 

* > •* • ♦ , - , * ' 



Figure 3. Human Fibroblast Cells Grown in the Presence of a One Tesla Magnet for 
Approximately 36 Hours